Chromatin structure and architecture.[unreadable] [unreadable] We are interested in the biophysical properties and structure of native chromatin fragments. The chicken folate receptor and beta-globin gene loci are ideal for such structural studies in that (i) the region possesses both condensed and transcriptionally active chromatin and (ii) the system has been extensively studied in terms of gene regulation, allowing us to relate the overall chromatin structure to transcription. The constitutively condensed chromatin region, spanning 15.5 Kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes, can be released from the cell nucleus with the restriction enzyme HpaII. We have previously analyzed the hydrodynamic properties of this condensed chromatin fragment and showed that it is an extended rod. This provides insights into the structure of heterochromatin, found interspersed within various genomes.[unreadable] [unreadable] The biophysical and biological tools developed in these studies have allowed us to further expand our investigation. Using an erythroid precursor cell line (6C2), we have shown that a 16.2 Kbp region of the beta-globin gene locus can be released from the nucleus with the restriction enzymes NheI and XhoI. In this particular cell line, this chromatin region possesses all the hallmarks of transcriptional inactivity. We have analyzed the hydrodynamic properties of this facultative heterochromatin fragment and showed that it is also an extended rod. Unlike the 15.5 Kbp constitutively condensed chromatin, however, this 16.2 Kbp chromatin fragment contains a smaller histone protein to nucleic acid ratio. Therefore, the similarity in structure observed highlights the flexibility of the chromatin structure in accommodating different DNA linker lengths. We have also studied the properties of the 15.5 and 16.2 Kbp chromatin fragments at higher ionic strengths and found no evidence of any abrupt conformational change, demonstrating that these chromatin fragments released from the nucleus did not assume the more compact conformations recently described for some reconstituted structures (Ghirlando and Felsenfeld,J. Mol. Biol.).[unreadable] [unreadable] We have further dissected the beta-globin gene cluster into a series of five distinct chromatin fragments ranging from 2.1 to 8.0 Kbp in size using the restriction enzyme BamHI. We showed that the dependence of the sedimentation coefficient with size is consistent with the extended rod like properties observed for chromatin. We are currently developing protocols to compare beta-globin genes chromatin fragments obtained from 6C2 cells with those released from 10-day old and adult chicken erythrocytes. As the beta-globin genes are transcribed in 10-day old erythrocytes but inactive in adult erythrocytes, these studies will allow us to relate the structures of constitutive and facultative heterochromatin with those of transcriptionally active and inactive chromatin.[unreadable] [unreadable] [unreadable] Macromolecular assemblies.[unreadable] [unreadable] In collaboration with members of the Laboratory of Molecular Biology, and other laboratories, protein and protein-nucleic acid assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies extend the biochemical and structural investigations and provide important mechanistic information. A case in point is provided by the recently published work on the complete yeast ESCRT-I heterotetramer carried out in collaboration with Dr. James H. Hurley. The endosomal sorting complex required for transport-I (ESCRT-I) complex is conserved from yeast to humans and directs the lysosomal degradation of ubiquitinated transmembrane proteins and, in humans, the budding of the human immunodeficiency virus (HIV). The complex is composed of Vps23, Vps28, Vps37, and Mvb12 and we show that these proteins assemble with high affinity to form an elongated and monodisperse 1:1:1:1 complex. Sedimentation data for the core complex are consistent with the structural data, and hydrodynamic studies on extended ESCRT-I constructs allow for a modeling of the intact ESCRT-I complex and a mechanistic understanding of how the complex interacts with the endosomal membrane. The elongated shape and dimension of approximately 25 nm indicate that the ESCRT-I complex participates directly in regulating the mechanical aspects of cargo recruitment and membrane remodeling (Kostelansky et al., 2007).